A monoclonal antibody against human vitamin-D-binding protein for the analysis of genetic variation in the group-specific component system (Gc).
We have developed a murine hybridoma cell line that is stable in secreting a monoclonal antibody (hDBP-1) directed against the group-specific component (Gc) molecule. The hDBP-1 is monospecific for Gc and does not crossreact with human albumin, which has 23% of its amino acid residues identical with vitamin-D-binding protein (DBP). The subclass of the antibody is IgG1 for the heavy chain, the light chain being of the kappa type. Isoelectric focusing discloses four major bands for the hDBP-1 with isoelectric points between pH 6.5 and 7.8. Binding to the antigen at different pH values was determined: there is high affinity in the physiological range and no binding at pH 3.5 and lower. In the presence of high salt concentrations, binding was reduced to about 50% at 1.5 M NaCl. The hDBP-1 recognizes the common human Gc types and the Gc of all apes and old world monkeys. No reaction was observed with the Gc of other mammals such as horses, cattle, rats, rabbits, sheep, goats and pigs. By testing hDBP-1 against 77 of the more than 120 known rare human Gc variants, it could be shown that this monoclonal antibody cannot recognize seven of these rare variants and can only poorly recognize nine. The binding site of hDBP-1 to Gc is not related to the binding site of Gc with G-actin: it recognizes Gc, the binary complex between Gc and G-actin, as well as the ternary complex between Gc, G-actin and DNase I. Competition assays with vitamin D3 and Gc in enzyme-linked immunosorbent assay indicate that the epitope of hDBP-1 on the Gc molecule may be related to the vitamin-D3-binding site.
Hoffmann
R
R
Institut für anthropologie und Humangenetik der Universität, München, Federal Republic of Germany.
Braun
A
A
Cleve
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D006801:Humans; D006825:Hybridomas; D007525:Isoelectric Focusing; D051379:Mice; D014809:Vitamin D-Binding Protein / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D014809:Vitamin D-Binding Protein